Fibronectin is a high molecular weight glycoprotein present in plasma and in extracellular matrices. Major functions of this protein include its role as a mediator of cellular adhesion and its affinity for various macromolecules. Fibronectin also enhances phagocytosis and promotes cell migration. These functions are located in structural domains which retain their activity following proteolytic digestion of the molecule. The purpose of this investigation is to contribute to the understanding of the mechanisms by which fibronectin performs its biological activities. The relation structure-function will be established by determining the primary structure of the active domains. In particular the fibrin- and heparin-binding regions will be studied. They will be isolated after subtilisin digestion of the molecule and appropriate affinity columns; further purification will be achieved by standard methods including HPLC. Purified fragments will be cleaved into smaller peptides suitable for amino acid analysis and amino acid sequence determination. Other properties which will also be studied are the binding of fibronectin to the P component of amyloid, the interaction with myeloma IgG and the enhancement of phagocytosis. The specific sites carrying these activities will be identified and their amino acid sequence determined. The second part of this application deals with the heterogeneity of the different forms of fibronectin. The recent demonstration of more than one fibronectin mRNA in human and rat fibroblasts and the different size of the two fibronectin chains strongly suggests the existence of several fibronectin molecules and raises questions regarding their origin and distribution. The approach that will be used is the amino acid sequence determination of proteolytic fragments derived from the regions containing differences between the two chains. A 140-kDa covalently bound fragment obtained after cathepsin D digestion seems most appropriate since it contains polypeptides from both chains. Other fragments from this region obtained by trypsin digestion will also be studied. Antisera prepared against peptides found exclusively in one chain may provide information on the distribution of the different forms of fibronectin. Similar studies will be performed on fibronectin isolated from cultured fibroblasts and the proteolytic products of both will be compared.